Microfabricated Channel Array Electrophoresis for Characterization and Screening of Enzymes Using RGS-G Protein Interactions as a Model System

Jian Pei; John F DISHINGER; David L ROMAN; Chetwana RUNGWANITCHA; Richard R NEUBIG; Robert T KENNEDY

doi:10.1021/ac800553g

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Microfabricated Channel Array Electrophoresis for Characterization and Screening of Enzymes Using RGS-G Protein Interactions as a Model System

Journal article

Peer reviewed

Microfabricated Channel Array Electrophoresis for Characterization and Screening of Enzymes Using RGS-G Protein Interactions as a Model System

Jian Pei, John F DISHINGER, David L ROMAN, Chetwana RUNGWANITCHA, Richard R NEUBIG and Robert T KENNEDY

Analytical chemistry (Washington), Vol.80(13), pp.5225-5231

2008

DOI: 10.1021/ac800553g

PMID: 18465881

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Abstract

A microfluidic chip consisting of parallel channels designed for rapid electrophoretic enzyme assays was developed. Radial arrangement of channels and a common waste channel allowed chips with 16 and 36 electrophoresis units to be fabricated on a 7.62 × 7.62 cm2 glass substrate. Fluorescence detection was achieved using a Xe arc lamp source and commercial charge-coupled device (CCD) camera to image migrating analyte zones in individual channels. Chip performance was evaluated by performing electrophoretic assays for G protein GTPase activity on chip using BODIPY−GTP as enzyme substrate. A 16-channel design proved to be useful in extracting kinetic information by allowing serial electrophoretic assays from 16 different enzyme reaction mixtures at 20 s intervals in parallel. This system was used to rapidly determine enzyme concentrations, optimal enzymatic reaction conditions, and Michaelis−Menten constants. A chip with 36 channels was used for screening for modulators of the G protein−RGS protein interaction by assaying the amount of product formed in enzyme reaction mixtures that contained test compounds. Thirty-six electrophoretic assays were performed in 30 s suggesting the potential throughput up to 4320 assays/h with appropriate sample handling procedures. Both designs showed excellent reproducibility of peak migration time and peak area. Relative standard deviations of normalized peak area of enzymatic product BODIPY−GDP were 5% and 11%, respectively, in the 16- and 36-channel designs.

Analytical Chemistry

Applied Sciences

Chemistry

Pollution

Chromatographic methods and physical methods associated with chromatography

Analysis methods

Exact sciences and technology

Wastes

Other chromatographic methods

Details

Title: Subtitle: Microfabricated Channel Array Electrophoresis for Characterization and Screening of Enzymes Using RGS-G Protein Interactions as a Model System
Creators: Jian Pei - Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055, United States
John F DISHINGER - Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055, United States
David L ROMAN - Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109-1055, United States
Chetwana RUNGWANITCHA - Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055, United States
Richard R NEUBIG - Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109-1055, United States
Robert T KENNEDY - Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055, United States
Resource Type: Journal article
Publication Details: Analytical chemistry (Washington), Vol.80(13), pp.5225-5231
DOI: 10.1021/ac800553g
PMID: 18465881
NLM abbreviation: Anal Chem
ISSN: 0003-2700
eISSN: 1520-6882
Publisher: American Chemical Society; Washington, DC
Language: English
Date published: 2008
Academic Unit: Pharmacy; Iowa Neuroscience Institute; Pharmaceutical Sciences and Experimental Therapeutics; Medicinal and Natural Products Chemistry
Record Identifier: 9984065494602771

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